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United States Patent |
5,231,025
|
Gralnick
|
July 27, 1993
|
Anti-platelet monoclonal antibody
Abstract
A unique anti-platelet monoclonal antibody which binds to human platelet
glycoprotein IV, and various utilities of the monoclonal antibody are
described.
Inventors:
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Gralnick; Harvey R. (Kensington, MD)
|
Assignee:
|
The United States of America as represented by the Department of Health (Washington, DC)
|
Appl. No.:
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432126 |
Filed:
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November 3, 1989 |
Current U.S. Class: |
435/337; 530/388.1; 530/388.25 |
Intern'l Class: |
C12N 005/12; C07K 015/28 |
Field of Search: |
530/387,413,388.25,413,415,388.1,388.25
435/240.27,172.2,70.21
935/104
424/85.8
|
References Cited
Other References
Sofer, G. et al., Biotechniques, 1983 Nov./Dec. pp. 198-203.
Kambayashi, H. et al., Leucocyte Typing III ed. by A. J. McMichael, p. 787,
1987.
|
Primary Examiner: Lacey; David L.
Assistant Examiner: Futrovsky; Susan L.
Attorney, Agent or Firm: Birch, Stewart, Kolasch & Birch
Claims
What is claimed is:
1. The hybridoma having ATCC deposit number HB10272.
2. The antibody secreted by the hybridoma identified as ATCC HB10272.
3. A monoclonal antibody of claim 2 which specifically binds to platelet
glycoprotein IV having the following defining characteristics:
1) Type IgG, subclass IgG1;
2) having approximately 11,000 specific binding sites per platelet cell;
3) specifically binding to a protein expressed on the surface of platelets,
said protein having a molecular weight of 88 kilodaltons when determined
under either reducing or non-reducing conditions;
4) specifically binding to platelet glycoprotein IV and not binding to the
platelet glycoprotein IIb/IIIa complex;
5) in the presence of plasma proteins, binding of said monoclonal antibody
induces platelet aggregation and causes the release of internal platelet
organelles;
6) causes platelet aggregation in a calcium-dependent,
magnesium-independent manner;
7) causes platelet aggregation independent of aspirin treatment of the
platelets;
8) causes platelet aggregation independent of the presence of adenosine
diphosphate or other typical platelet aggregation agonists; and
9) induces the binding of adhesive proteins, such as fibrinogen and Von
Willebrand factor, to the surface of platelets.
4. A composition of matter, comprising an effective amount of the antibody
of claim 2 to induce platelet aggregation or to bind with GPIV, and a
carrier.
Description
The present invention is related generally to monoclonal antibodies. More
particularly, the present invention is related to a unique monoclonal
antibody (Mab) against resting human platelets, said Mab being designated
herein as 5G8 and which recognizes a human platelet surface protein of
about 88kD, in both reduced and nonreduced state. Such a monoclonal
antibody has not heretofore been known or described.
BRIEF DESCRIPTION OF THE DRAWINGS
Various features and attendant advantages of the invention will be better
understood upon a reading of the following detailed description when
considered in connection with the accompanying drawings wherein:
FIG. 1 demonstrates the platelet surface glycoprotein which is precipitated
by the antibody 5G8 in a 5% polyacrylamide gel in the presence of SDS in a
nonreduced state, however identical results were obtained when the
antigens were reduced (88kD). Demonstrated in lane 1 is the
immunoprecipitate of an antibody against GMP140. In lane 3 is the
immunoprecipitate by another antibody 76D which recognizes the
Glycoprotein Ia/II complex, molecular weight 150 and 135kD. In lane 2 the
results indicate that the 5G8 antibody immunoprecipitates a surface
protein that has a molecular weight of 88kD.
FIG. 2H-2F shows the dose response curve of the 5G8 antibody added to
normal platelet-rich plasma. FIG. 2A platelet-rich plasma without the
addition of monoclonal antibody 5G8, FIG. 2B, addition of 1 .mu.g/ml 5G8,
FIG. 2C addition of 5 .mu.g/ml, FIG. 2D, addition of 10 .mu.g/ml, FIG. 2E
addition of 20 .mu.g/ml, and FIG. 2F addition of 40 .mu.g/ml. Normal
platelet-rich plasma by itself did not aggregate; however, after the
addition of 185G8 from 1 to 40 micrograms/ml final concentration, the
platelet aggregation ensued. There was a clear dose response curve between
the amount of 5G8 added and the initial slope and total amount of platelet
aggregation.
FIG. 3 demonstrates that the antibody of the present invention increases
the binding of fibronectin, fibrinogen, or von Willebrand factor to
unstimulated platelets (FIG. 3a). This was related to the amount of
antibody present. When the antibody was incubated with the ligand rather
than with the platelets, this had no effect on the binding of the ligand
to platelets. Lower concentrations of 5G8 can enhance binding of ligands
to platelets that have been minimally stimulated. The largest increase in
binding was observed with either ADP or epinephrine as agonists and
fibrinogen 3b or von Willebrand factor 3c as the ligands. The agonist is 2
.mu.M ADP. The antibody can also enhance the binding of fibrinogen, von
Willebrand factor and fibronectin to platelets which had been previously
stimulated with thrombin. The greatest enhancement of binding was observed
with the agonist ADP and epinephrine. Note that at 0.1 .mu.g/ml 5G8
increases the binding of fibrinogen 12 fold over control (no antibody
present) binding, and 5G8 at 0.1 .mu.g/ml increases von Willebrand factor
4 fold over control. In FIG. 3a 0.1 .mu.g/ml has no effect on fibrinogen
or von Willebrand factor binding.
DETAILED DESCRIPTION OF THE INVENTION
Various objects and advantages of the present invention are achieved by
producing monoclonal antibody 5G8 which, inter alia, has the following
properties.
(a) Belongs to subclass IgG1.
(b) Binds to about 11,000 sites per platelet with a platelet surface
protein of about 88kD in reduced or nonreduced form (FIG. 1).
(c) Induces spontaneous platelet aggregation in the presence of plasma
proteins, such as when added to whole blood or to normal platelet rich
plasma, causing release of internal platelet organelles and formation of
large platelet aggregates (FIG. 2).
(d) Distinctive from other platelet related MAbs, the MAb specifically
recognizes glycoprotein IV (GPIV) and modulates platelet aggregation by
binding to an epitope on GPIV and does not recognize GP IIb/IIIa complex.
(e) Platelet aggregation induced by this MAb is calcium dependent, but does
not require magnesium and is not affected by aspirin treatment of
platelets or by released ADP, nor other agonists are required for
aggregation reaction to take place.
Unless defined otherwise, all technical and scientific terms used herein
have the same meaning as commonly understood by one of ordinary skill in
the art to which this invention belongs. Although any methods and
materials similar or equivalent to those described herein can be used in
the practice or testing of the present invention, the preferred materials
and methods are now described. All publications mentioned hereunder are
incorporated herein by reference. Unless mentioned otherwise, the
techniques employed or contemplated herein are standard methodologies well
known to one of ordinary skill in the art. The materials, methods and
examples are illustrative only and not limiting.
The term "substantially pure" as used herein means as pure as can be
obtained by conventional purification and isolation techniques well known
to one of ordinary skill in the art.
MATERIALS AND METHODS
The immunogen used for production of the monoclonal antibody 5G8 was
stimulated platelets that had been formalin fixed after 5 minutes
incubation with thrombin. Before stimulation and fixation the platelets
were purified from other cells and plasma proteins by centrifugation on
arabinogalactan gradient (5 ml of 10% and 3 ml of 20%). The platelets were
prepared from whole blood by centrifugation at 600 g for 3 minutes at room
temperature (about 22.degree.-24.degree. C.). Platelet-rich plasma was
removed and was separated from plasma proteins and lymphocytes on an
arabinogalactan gradient. These platelets, free of other cellular
contamination, were then activated with thrombin 0.1 u/ml, final
concentration, per 1.times.10.sup.8 platelets. After about 5 minutes, the
platelets were mixed with equal volume of 2% formalin. Three injections of
these cells were made into BALBc mice prior to the fusion (vide infra).
The fusion was performed with PU mouse myeloma cells and murine
splenocytes using polyethylene glycol. At 7-10 days, the wells were
inspected for growth positivity. Supernatants were screened for antibody
production by standard ELISA technique and by cytometry. The positive
individual wells selected by the screening procedures were then expanded
to 24 macrowell plates containing H-T media and murine spleen feeder
cells. When supernatant from the macrowells were tested and found to be
positive, these clones were then subjected to limiting dilution studies,
the 5G8 underwent 6 limiting dilutions. The fused cells were then injected
intraperitoneally in pristine BALB/c mice, and ascites fluid was collected
after 2-3 weeks. The antibody was purified by chromatography over a
protein-A Sepharose column. The antibody, 5G8, was found to belong to Ig1
class.
FUSION PROCEDURE
1. Seven days before fusion, pass PU mouse myeloma cells at
3.5.times.10.sup.5 /ml in complete media with 1% 8-azaguanine (10- 20
g/ml) total volume 100 ml. On the fifth day after addition of
8-azaguanine, add 100 ml complete media to culture. On the sixth day,
centrifuge cells 100 rpm 10.sup.1, and resuspend in complete media at a
concentration of 3.5.times.10.sup.5 cells/ml. On the seventh day cells
should be in mid log phase 4-6.times.10.sup.5 /ml. For each fusion, take
3.times.10.sup.7 PU cells and wash in RPMI 1640 with glutamine,
Penicillin-streptomycin (pen-strep). (Note no FCS at this step). Resuspend
in 1 ml RPMI with glutamine, penstrep.
2. Autoclave PEG for 15'. Dilute v/v with RPMI (Glutamine, pen-strep) and
add 1 drop sterile 7.5% sodium bicarbonate for each ml of 50% PEG.
3. Remove immunized mouse spleen aseptically and place in small petri dish
containing 8 ml RPMI+glutamine+2X pen-strep. Use small sterile syringe
plunger to homogenize spleen. Transfer spleen to 15 ml centrifuge tube and
rinse petri with 2 ml RMPI+glutamine+2X pen-strep. Let spleen settle 1-2'
to get rid of large debris. Remove 9.5 ml and add to washed
3.times.10.sup.7 PU cells. Spin at 1400 rpm for 10'. Aspirate off as much
supernatant as possible. Gently tap test tube over top of test tube rack
to spread out cell pellet. Add 1 ml 50% PEG at 37.degree. C. dropwise over
one minute to cell pellet. Add 5 ml RPMI (glutamine, pen-strep) over 5
minutes (add 1 ml in drops over 1 minute, then add 4 ml and wait 4
minutes). Fill tube with RPMI (glutamine, pen-strep) a second time and
centrifuge 1000 rpm 10'. Resuspend pellet in 100 ml selective cloning
media and place in 5 microtiter 96-well flat bottom plates. Cover/wrap
with parafilm and plate in 37.degree. C., 7% CO.sub.2 incubation.
4. At 7-10 days inspect wells for growth positivity and acidification of
media (yellowing of media). When positive, supernatants must be screened
for antibody production and/or antibody specificity by standard ELISA
protocol.
5. Individual wells selected by screening procedure are then expanded to 24
macrowell plates containing H-T media and murine spleen feeder cells.
Ideally, a limiting dilution (LD) is to be carried out immediately at the
time of supernatant positivity; however, it can be carried out at the
macrowell step. Regardless of procedure followed, the parenteral "clone"
should be cryopreserved as soon as possible.
6. For LD, obtain cell count and viability on clone. Also prepare spleen
feeder cells (1 spleen for five plates suspended in 10 ml of HT as per
step 3). Essentially want 100 hybridoma cells per 96-well plate and
approximately 2.times.10.sup.5 spleen feeder cells/well. LD should be
carried out .times.3 to ensure clonal origin of hybridoma. Subclones from
each LD and final clones should be cryopreserved.
Complete Media
RPMI 1640: Biofluids Cat. #102
50 ml Fetal calf serum 309 (heat inactivated): Gibco Cat. #240-6309
10 ml L-glutamine: Gibco Cat. #320-5030
0.5 ml Gentamicin: Gibco Cat. #600-5710.
RPMI 1640
Biofluids VCat. #102
10 ml L-glutamine: Gibco Cat. #320-5030
5.0 ml Penicillin-Streptomycin mixture:
M.A. Bioproducts Cat. #17-6034.
CDME
450 ml DMEM: Biofluids Cat. #104
0.474 ml Penicillin-streptomycin mixture: M.A. Bioproducts Cat. #17-6034
9.47 ml 200 mM L-glutamine: Gibco Cat. #320-5030
59.3 ml FCS (heat inactivated): Gibco Cat. #240-6309
4.74 ml Nonessential amino acids: M.A. Bioproducts Cat. #173-114A
2.84 ml Sodium pyruvate: Gibco Cat. #320-1360
59.3 ml NCTC 109: Gibco Cat. #320-1340.
Selective cloning media
99 ml CDME
1.0 ml HAT (hypoxanthine, aminopterin, thymidine): Hazelton Cat. #59-77076.
PEG 1500 (polyethylene glycol)
M.A. Bioproducts, Cat. #17-7802
HT (Hypoxanthine, Thymidine)
Hazelton Cat. #59-57076
99 ml CDME
1.0 ml HT.
Costar 96-2311 flat-bottom tissue culture plates
Cat. #3596
A deposit of the hybridoma secreting MAb 5G8 has been made at the American
Typeculture Collection, 12301 Parklawn Drive, Rockville, Md., 20852, on
Oct. 24, 1989, under accession number HB10272. The deposit shall be viably
maintained, replacing if it becomes non-viable during the life of the
patent, for a period of 30 years from the date of the deposit, or for 5
years from the last date of request for a sample of the deposit, whichever
is longer, and upon issuance of the patent made available to the public
without restriction in accordance with the provisions of the law. The
Commissioner of Patents and Trademarks, upon request, shall have access to
the deposit.
Specificity of 5G8
Data presented in FIGS. 1-3 provide evidence of various properties of 5G8
listed herein above.
Utility of 5G8
The application of 5G8 include its utility in identifying a specific
platelet defect in individuals with coagulation or hemostatic disorders.
This can be done by testing this antibody to see if it enhances platelet
aggregation and ligand binding to platelets in certain disease state. This
antibody could also be used as an antihemorrhagic agent in which injection
of this antibody would enhance platelet attachment and platelet
aggregation at sites of vessel wall injury. It would be a useful aid in
stopping or reducing surgical bleeding and enhancing normal wound healing.
It likewise would be used not only by intravenous injections but could be
used topically for a variety of minor cuts, scrapes, bruises, etc., to
augment platelet plug formation and blood clotting at that site. A
necessity to decrease microvascular bleeding may arise either from surgery
or by trauma and 5G8 could be used locally or systemically to augment
subnormal or normal mechanisms of hemostasis. It can also be used as a
reagent for the purification of GPIV from platelets. This involves the
attachment of antibody 5G8 to a semi-solid surface (such as agarose) and
to passing normal platelet membranes over this antibody affinity column.
The GPIV would be selected (attached) to the antibody and eluted from the
antibody. This antibody further allows investigation of the role(s) of
GPIV in normal and pathological platelet function. It is not known what
pathway(s) of platelet activation and aggregation are involved. New
insights into these processes are important in thrombosis and hemostasis.
The availability of the anti-platelet MAb 5G8 also allows the preparation
of antihemorrhagic composition containing an effective amount of 5G8 to
react with GPIV, and a pharmaceutically acceptable carrier well known to
one of ordinary skill in the art, such as physiological saline, nontoxic
sterile buffer and the like. A kit comprises a container containing the
antibody 5G8, either cryopreserved or otherwise.
It is understood that the examples and embodiments described herein are for
illustrative purposes only and that various modifications or changes in
light thereof will be suggested to persons skilled in the art and are to
be included within the spirit and purview of this application and scope of
the appended claims.
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